Our research program is designed to study the synthesis, assembly, structure and function of the proton-translocating ATPase (F1Fo) of E. coli. This important enzyme complex is the central component in the utilization of an electrochemical gradient of protons to synthesize ATP (oxidative phosphorylation) and to hydrolyse ATP to generate an electrochemical gradient of protons essential for other energy dependent process of the cell. We plan to study the assembly of the polypeptides of the complex with regard to each other and the cytoplasmic membrane. We will determine the association state of the three Fo peptides, a, c and b, in their functional and non-functional states using proteolysis, cross-linking and labelling reagents. We will determine the role of the F1 subunits, Alpha and Beta in Fo assembly. We will determine the assembly sequence of the 5 F1 polypeptides, Alpha, Beta, Gamma, Epsilon, and Sigma. We will assemble the Fo portion of the complex into black lipid membranes in order to study the biogenesis process in vitro and to study the proton conduction mechanism. We plan extensive genetic analysis of the complex in order to understand the role of each of the polypeptides in the overall structure, assembly and function of the complex. We will focus primarily on in vitro mutagenesis of the uncF(b), uncB(a) and uncH(Delta) genes coupled with a thorough biochemical characterization of the consequences of each mutation. We will also conduct a systematic intercistronic complementation analysis within the unc operon. Attempting, by genetic means, to establish subunit interactions particularly polypeptide domains which interact. We will also conduct a detailed genetic/physiological analysis of the uncI gene and a biochemical analysis of the function of the uncI gene product, i, in an attempt to determine if this gene and protein are essential for function of the complex, and if so, in what way.